1. Field of the Invention
The present invention relates to a method and an apparatus for identifying substances by measuring the energy transferred between and within molecules and more particularly to detecting small amounts of substances within living cells by measuring energy transfer.
2. Description of the Related Art
Fluorescence resonance energy transfer (FRET) within or between two different types of molecule, that occurs when energy from an excited donor fluorophore is transferred directly to an acceptor fluorophore, is a useful phenomenon for studying the character of molecules. This method is especially useful for in vitro measurements of small quantities of substances and can be applied to analysis of genetic information to measure expression of genes and changes in the primary structure of DNA and RNA to a high degree of precision.
Next, an explanation will be provided for a general method for measuring energy transfer from an excited fluorophore (donor) to an absorber (acceptor).
1. The spectra, including changes in the spectra, of fluorescence from the donor and acceptor are measured.
2. Reduction in intensity of fluorescence from the donor or increase in intensity of fluorescence from the acceptor is measured.
3. The speed at which the intensity of the fluorescent intensity of the donor decreases after pulse-laser excitation (i.e., the fluorescence lifetime) is measured.
However, sometimes the sample contains numerically more molecules that do not emit energy (i.e., free molecules) than molecules that do emit energy, in which case measurement using this three-step method is impossible. This three-step method is also not possible when the density of energy-transferring donors or acceptors can not be determined. Because in this three-step method fluorescence from both energy-emitting and non-energy emitting molecules is measured, the characteristic change in fluorescence which occurs from energy transfer is buried in the fluorescence produced by molecules that do not emit energy. Also, when the increase in fluorescence intensity in the acceptor is measured in step 2, the acceptor directly absorbs some of the excitation light and fluoresces at an intensity significant compared to the intensity of acceptor fluorophore emission from energy transfer. This makes determination of only the energy transfer induced fluorescence from the acceptor fluorophore impossible.
Larry E. Morrison describes a method of measuring energy transfer under these conditions (Analytical Biochemistry 174, pp 101-120, 1988). His technique "requires the selection of donor and acceptor fluorophores such that the fluorescence lifetime of the donor is greater than the fluorescence lifetime of the acceptor." The fluorescence emitted from the acceptor is measured a predetermined duration of time (set by a delay gate) after the donor fluorophore has been excited by the pulse of light. With this method, the fluorescence emitted from the acceptor as a result of direct absorption of the excitation light is temporally separated from the fluorescence emitted from the acceptor by energy transfer. Measurement of energy transfer is improved because the fluorescence contributed by light excitation (i.e., not by energy transfer) of the acceptor is eliminated.
Roger Y. Tsein et al. describe a method wherein the ratio of the fluorescent intensities of the donor and the acceptor when excited by a certain wavelength excitation light is calculated and an image produced from the results (Trends in Cell Biology, Vol. 3, pp. 242-245, 1993). Takatoku Oida et al. describe temporal analysis of imaging (Biophys. J., Vol. 64, pp.676-685, 1993). With these two methods, fluorescence for each fluorophore can be separated by electric gating of detector signals. Scattering of light or mixing with light other than the objective fluorescence can be prevented. Precision in measuring fluorescent intensity can be increased by differences in the length of the optical pathways. Influence of unknown densities of molecules can also be reduced. Energy transfer within cultivated cells can be measured under a microscope.